| RNAi has been widely used to block or suppress gene expression due to its sequence-specific advantages,and has become a powerful tool for research and development of transgenic plants.However,there is not has not a safety evaluation guide specifically for RNAi transgenic plants in CHINA.To provide theoretical support for the refinement of the indicators of environmental safety assessment of RNAi transgenic plants,this study took the specific expression of the seed-specific NAPIN promoter-driven fad2-RNAi high oleic acid transgenic rape W-4 and wild-type Westar of Brassica napus as research objects.The molecular characteristics,genetic stability,gene expression and metabolic product difference analysis of the transgenic material were studied,and the difference characteristics between the RNAi transgenic plant and the wild type were comprehensively compared,and the following results were obtained:(1)Through genome-wide resequencing combined with PCR verification,the insertion site of the foreign vector in W-4 was determined to be integrated on the right side of the Brassica napus genomic database ChrA09.26345542-26345904;the left side is integrated into the Brassica napus genome database ChrA09-random.3011655-3012135 and verify the integrity of the transferred sequence,and the number of genome-wide mutation sites and chromosome distribution characteristics of T3 and T9 transgenic lines of W-4 were analyzed.(2)The real-time fluorescence quantitative PCR confirmed that the transcriptional expression of the fad2 gene was significantly lower than that of wild-type Westar in the seeds of transgenic lines of more than four generations of W-4.The expression of FAD2 protein was detected using specific oilseed rape FAD2 antibody,and it was found that the expression of FAD2 protein in the mature leaves of Fad2-RNAi high oleic acid transgenic rape W-4 was consistent with the wild-type material;the mature pods 21 days after flowering were activated by napin.The seed has been expressed and fad2 transcription is suppressed,so the expression of FAD2 protein in wild-type WT mature pods is higher than that of Fad2-RNAi high oleic acid transgenic rape W-4.(3)Through extensive targeted metabolomics analysis,112 different metabolites were found between W-4 and wild type.24 kinds of down regulated differential metabolites were detected,10 types of phenylpropanoids,3 types of lipids,3 types of flavonols,2 types of polyphenols,2 types of organic acids and derivatives,amino acids and Derivatives,phenolamines,nucleotides and derivatives,vitamins and derivatives,each one.88 up-regulated differential metabolites were detected,19 amino acids and derivatives,15 organic acids and derivatives,14 lipids,6 nucleotides and derivatives,and 5 flavanones There are 4 types of alkaloids,3 types of phenylpropanoids,flavonoids,and flavonols,2 types of alcohols,polyphenols,and procyanidins,phenolamines,terpenes,vitamins and derivatives There are 1 species of indole and 1derivative,and 6 species of others.Further analysis of the transcriptional expression of key genes in related metabolic pathways by real-time quantitative PCR revealed that the lpat2,dgat2,and fad3 genes in W-4 seeds were up-regulated relative to the wild type in all generations of transgenic plants compared to wild type;The tgd1-5 gene was down-regulated relative to the wild type in all generations of transgenic plants.Based on the above research results,this paper clarifies the insertion site and system of the transgenic sequence of transgenic rape W-4 and understands the genomic change characteristics,target gene expression change characteristics and metabolite change characteristics,and provides for the subsequent safety evaluation declaration of the GM material It not only provided experimental basis,but also provided theoretical basis and scientific data for the development of RNAi transgenic safety evaluation guidelines. |
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